The native prokaryotic CRISPR-Cas system comprises an array of short repeats with intervening variable sequences of constant length (i.e., clusters of regularly interspaced short palindromic repeats, or “CRISPR”), and CRISPR-associated (“Cas”) proteins. The RNA of the transcribed CRISPR array is processed by a subset of the Cas proteins into small guide RNAs, which generally have two components as discussed below. There are at least six different systems: Type I, Type II, Type III, Type IV, Type V and Type VI. The enzymes involved in the processing of the RNA into mature crRNA are different in the six systems. In the native prokaryotic Type II system, the guide RNA (“gRNA”) comprises two short, non-coding RNA species referred to as CRISPR RNA (“crRNA”) and trans-acting RNA (“tracrRNA”). In an exemplary system, the gRNA forms a complex with a Cas nuclease. The gRNA:Cas nuclease complex binds a target polynucleotide sequence having a protospacer adjacent motif (“PAM”) and a protospacer, which is a sequence complementary to a portion of the gRNA. The recognition and binding of the target polynucleotide by the gRNA:Cas nuclease complex induces cleavage of the target polynucleotide. The native CRISPR-Cas system functions as an immune system in prokaryotes, where gRNA:Cas nuclease complexes recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms, thereby conferring resistance to exogenous genetic elements such as plasmids and phages.
It has been demonstrated that a single-guide RNA (“sgRNA”) where the crRNA and tracrRNA are covenlently linked can replace the complex formed between the naturally-existing crRNA and tracrRNA.
Considerations relevant to developing a gRNA, including a sgRNA, include specificity, stability, and functionality. Specificity refers to the ability of a particular gRNA:Cas nuclease complex to bind, nick, and/or cleave a desired target sequence, whereas less or no binding, nicking, and/or cleavage of polynucleotides different in sequence and/or location from the desired target occurs. Thus, specificity refers to minimizing off-target effects of the gRNA:Cas nuclease complex. There is a need for providing gRNA, including sgRNA, having desired binding affinity for the target polynucleotide with reduced off-target effects while, nonetheless, having desired gRNA functionality. There is also a need for improved ways to make and use gRNA, including sgRNA, having enhanced specificity, with desired binding affinity for the target polynucleotide and/or reduced off-target binding.